Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 70 No. 6 pp. 951ñ959, 2013 ISSN 0001-6837 Polish Pharmaceutical Society

ANALYSIS

IDENTIFICATION AND DETERMINATION OF KETOTIFEN HYDROGEN FUMARATE, AZELASTINE HYDROCHLORIDE, DIMETINDENE MALEATE AND PROMETHAZINE HYDROCHLORIDE BY DENSITOMETRIC METHOD

ELØBIETA WYSZOMIRSKA1*, KRYSTYNA CZERWI—SKA1, ELØBIETA KUBLIN1 and ALEKSANDER P. MAZUREK1,2

1Department of Basic and Applied Pharmacy, National Medicines Institute, 30/34 Che≥mska St. 00-725 Warszawa, Poland 2Department of Drug Chemistry, Medical University of Warsaw, 1 Banacha St., 02-097 Warszawa, Poland

Abstract: Conditions for determination of: ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate and promethazine hydrochloride by densitometric method in substances and pharmaceuticals were pro- vided. Maximum wavelenghts were: 228 nm for ketotifen hydrogen fumarate, 295 nm for azelastine hydrochlo- ride, 265 nm for dimetindene maleate and 255 nm for promethazine hydrochloride. The limits of quantification were in the ranges of 0.2ñ5 µg/spot. The statistical data showed adequate accuracy and precision of developed methods.

Keywords: ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate, promethazine hydrochloride, histamine antagonist, densitometry

Allergy means all excessive reactions of healthy people to neutral substances. Histamine is a physiologically active, endoge- nous substance that activates H1 and H2 receptors. It is responsible for allergic reaction in our body. The mechanism of action of antihistamines is based on their competitive and reversible connection between H1 and H2 receptors. It annihilates effects of C23H23NO5 S M.w. 425.5 histamine as a mediator of inflammation and Ketotifen hydrogen fumarate - 4-(1-Methylpiperidin-4-ylidene)- immune response (1, 2). 4,9-dihydro-10H-benzo[4,5]cyclohepta[1,2-b]thiophen-10-one Ketotifen hydrogen fumarate, azelastine hydrogen (E)-butenedioate hydrochloride, dimetindene maleate and promet- hazine hydrochloride are histamine antagonists Azelastine (as hydrochloride) is a II genera- medicines usually used in allergy treatment. tion antihistamine drug. Not only does it block

Therefore, these medicines were chosen for this receptors for H1, but also inhibits the release of his- study. tamine. Following oral administration, it is metab- Ketotifen (as hydrogen fumarate) is able to olized and a major metabolite, which is N- inhibit the release of histamine, other relay demethylazelastine, shows properties of antihista- basophils and mast cells. It causes long-lasting inhi- mines. It is used in symptomatic treatment of aller- bition of histamine reactions. It is effective in the gies and as an adjunct in treatment of bronchial treatment of allergic diseases as well as asthma. It is asthma (1). used to prevent asthma attacks caused by allergy, it also works antianafilactically (1, 2).

* Corresponding author: e-mail: [email protected]

951 952 ELØBIETA WYSZOMIRSKA et al.

chemiluminescence method (3) and in plasma using HPLC method with a mass spectrometry detector (4). Azelastine, its enantiomers and metabolites were determined in rats plasma using coupling HPLC method with a β-cyclodextrin chiral sta- tionary phase to ion-spray tandem mass spectrom- etry. Additionally, an enatioselective assay was compared to another assay using electrokinetic capillary chromatography with β-cyclodextrin C H Cl N O M.w. 418.4 22 25 2 3 β Azelastine hydrochloride - 4-(4-Chlorobenzyl)-2-[(4RS)-1- and carboxymethyl- -cyclodextrin in polyacryl- methylhexahydro-1H-azepin-4-yl]phthalazin-1(2H)-one amide-coated capillaries (5). Moreover, HPLC hydrochloride method, with a mass spectrometry detector and chiral column, was used to determine azelastine in Dimetindene (as maleate) is a I generation plasma (6). antihistamine. It shows a very strong antipruritic Liquid chromatography (LC) with ZIC-HILIC activity. It is administered orally: retard tablet column and UV detector was used to determine (every 12 h) or drops and topically on the skin as a dimetindene maleate in gel (7). Capillary elec- gel (1, 2). trophoresies (CE) with a diode array detection (DAD) was used to determine dimetindene binding to human plasma proteins (8). Promethazine hydrochloride was determined in medicinal products (9ñ14) using spectrophotometric method (9, 10), capillary electrophoresis (11), den- sitometry (12), portable system of programmable syringe pump with potentiometer (13) and highly accurate nephelometric titration (14). Voltammetric method with carbon paste electrode (15) and HPLC- ESI-MS (16) were used to determine promethazine C H N O M. w. 408.5 24 28 2 4 in biological material. Dimethindene maleate - N,N-Dimethyl-2-[3-[(RS)-1-(pyridin-2- yl)ethyl]-1H-inden-2-yl]ethanamine (Z)-butenedioate Due to a high incidence rate of allergic reac- tions, histamine antagonists are a widely used group of compounds. Cost effective and easy methods for Promethazine (as hydrochloride) is a phenothi- the determination are required as an alternative to azine derivative of commonly used, slow, but long- liquid chromatography. acting antihistaminic. It also has hypnotic proper- The purpose of the study was to develop a sen- ties, antiemetic, anticholinergic and analgesic. sitive, simple, and cost-effective TLC method for Promethazine as therapeutic doesnít affect cardio- identification and determination of discussed com- vascular system, neither does it work as psychotic or pounds: ketotifen hydrogen fumarate, azelastine neuroleptic (1, 2). hydrochloride, dimetindene maleate and promet- hazine hydrochloride.

EXPERIMENTAL

Reference materials Ketotifen hydrogen fumarate (Novartis), Azelastine hydrochloride (Ph. Eur.), Dimetindene C17H21ClN2S M.w. 320.9 maleate (Ph. Eur.), Promethazine hydrochloride (Ph. Promethazine hydrochloride - (2RS)-N,N-Dimethyl-1-(10H-phe- nothiazin-10-yl)propan-2-amine hydrochloride Eur.).

Active substances From the literature on the subject it can be Ketotifen hydrogen fumarate (Novartis), concluded that during last years, ketotifen hydro- Azelastine hydrochloride (Meda Pharma), Dimetin- gen fumarate was determined in tablets using dene maleate (Novartis). Identification and determination of ketotifen hydrogen fumarate... 953

Medicinal products The following seven chromatographic systems were Ketotifen 1 mg tablets (Polfa Warszawa), used:

Allergodil 0.1% solution (Meda Pharma), Fenistil 1 1. Merck HPTLC Silica gel 60 F254 chromato- mg/g gel (Novartis), Diphergan 25 mg dragÈes graphic plates and mobile phase: trichloromethane - (Jelfa). methanol - ammonia 25% (90 : 10 : 1, v/v/v) with chamber saturation;

Reagents and apparatus 2. Merck HPTLC Silica gel 60 F254 chromato- Analytically pure and high purity reagents for graphic plates and mobile phase: diethylether ñ HPLC from Lab Scan, Merck HPTLC Silica gel 60 diethylamine (40 : 1, v/v);

F254 and Merck HPTLC LiChrospher Silica gel 60 3. Merck HPTLC Silica gel 60 F254 chromato-

RP-18 WF254s chromatographic glass plates, Hanau graphic plates and mobile phase: methanol - ammo- UV lamp, Camag automatic applicator, Shimadzu nia 25% (100 :1.5, v/v); CS 9000 densitometer, Dionex Ultimate 3000 liquid 4. Merck HPTLC LiChrospher Silica gel 60 chromatograph or Shimadzu liquid chromatograph RP-18 WF254s chromatographic plates and mobile with SPD-10 AVVP spectrophotometric detector, phase: acetonitrile ñ methanol - acetate buffer pH SCL-10 AVP autosampler and LC-10 AT VP pump. 5.5 (3 : 2 : 5 v/v/v);

5. Merck HPTLC Silica gel 60 F254 chromato- Qualitative analysis graphic plates and mobile phase: toluene - ethyl Standard solutions of ketotifen hydrogen acetate ñ acetic acid 100% (16 : 4 : 1, v/v/v); fumarate, azelastine hydrochloride, dimetindene 6. Merck HPTLC Silica gel 60 F254 chromato- maleate and promethazine hydrochloride in graphic plates and mobile phase: n-hexane ñ acetone methanol of the following concentrations: 1 mg/mL, ñ ammonia 25% (85 : 14 : 1, v/v/v);

0.1 mg/mL, 0.01 mg/mL were prepared. 7. Merck HPTLC Silica gel 60 F254 chromato- Quantities of 30, 20, 10, 5, 2.5, 2, 1.5, 1, 0.5, graphic plates and mobile phase: acetonitrile ñ water 0.25, 0.2, 0.1, 0.05 and 0.025 µg of active substances ñ ammonia 25% (90 : 10 : 1, v/v/v). were put as spots onto Merck HPTLC Silica gel 60 The plates were air dried and the spots posi-

F254 or Merck HPTLC LiChrospher Silica gel 60 RP- tions were determined under 254 nm UV light and in

18 WF254s chromatographic plates (2 cm from the iodine vapors. Rf values were determined. Limits of edge and 2 cm from the bottom) and developed in detection (LOD) were established for all analyzed selected mobile phases up to 1 cm from the top edge. compounds by visual methods.

Figure 1. UV spectrum of ketotifen hydrogen fumarate measured directly from the TLC plate 954 ELØBIETA WYSZOMIRSKA et al. chro- 254 2. Merck HPTLC lica gel 60 F chromatographic plates and methanol ñ ammonia 25% (100 : 1.5, v/v) mobile chromatographic plates and n-hexane ñ acetone ammonia 25% (85 : 14 1, 254 254 0 F ntitative determination of a given compound). s chromatographic plates and acetonitrile ñ methanol acetate buffer pH 5.5 (3 : 2 5, v/v/v) mobile phase; 5. Merck HPTLC Si chromatographic plates and trichloromethane ñ methanol ammonia 25% (90 : 10 1, v/v/v) mobile phase with chamber saturation; 254 254 chromatographic plates and acetonitrile ñ water ammonia 25% (90 : 10 1, v/v/v) mobile phase. 254 Limit of Limit of Limit of Limit of Limit of Limit of Limit of [µg/spot] [µg/spot] [µg/spot] [µg/spot] [µg/spot] [µg/spot] [µg/spot] UV iodine UV iodine UV iodine UV iodine UV iodine UV iodine UV iodine [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] [µg] System 1 System 2 System 3 System 4 System 5 System 6 System 7 0.54 0.20.64 0.1 0.05 0.280.96 0.1 0.1 0.1 0.28 0.5 0.02 0.1 0.49 0.1 0.44 0.51 0.1 0.05 0.1 0.05 - 0.5 0.1 0.62 0.1 0.26 0.1 0.1 0.1 0.25 0.2 0.09 0 0.1 0 0.1 0.25 0.1 0.06 0.1 0.1 0.2 0 0.25 0 0.25 0.16 0.25 0.1 0.1 0.36 0.1 0.25 0.2 0.6 0.26 0.1 0.15 0.1 0.25 0.2 chromatographic plates and diethylether ñ diethylamine (40 : 1, v/v) mobile phase; 3. Merck HPTLC Silica gel 60 F 254 maleate fumarate hydrogen hydrogen 0.96 0.25 0.1 0.34 0.25 - 0.58 0.25 0.25 0.15 0.1 0.25 0.02 0.25 0.25 0 0.25 0.25 0.5 0.25 0.25 Ketotifen Azelastine Dimetindene Promethazine hydrochloride hydrochloride Tested compoundTested Rf detection Rf detection Rf detection Rf detection Rf detection Rf detection Rf detection phase; 4. Merck HPTLC LiChrospher Silica gel 60 RP-18 WF v/v/v) mobile phase; 7. Merck HPTLC Silica gel 60 F Table 1. Rf values and limits of detection for the tested compounds (gray fields refer to mobile phase selected qua Chromatographic systems: 1. Merck HPTLC Silica gel 60 F matographic plates and toluene ñ ethyl acetate acetic acid 100% (16 : 4 1, v/v/v) mobile phase; 6. Merck HPTLC Silica gel 6 Silica gel 60 F Identification and determination of ketotifen hydrogen fumarate... 955

The results are presented in Table 1. matographic plates and mobile phase: methanol ñ ammonia 25% (100 : 1.5, v/v). Densitometric Densitometric quantitative analysis analyses were conducted using Shimadzu CS The following systems were used for deter- 9000 densitometer. Chromatographic plates were mination of analyzed compounds: system II for placed inside the chamber. Slit dimensions were azelastine hydrochloride, dimetindene maleate 0.4 ◊ 0.4 mm. Measurements were made by and promethazine hydrochloride: Merck HPTLC zigzag scanning with the following width of

Silica gel 60 F254 chromatographic plates and deflection: 10 mm for azelastine hydrochloride, mobile phase: diethylether ñ diethylamine (40 : 12 mm for ketotifen hydrogen fumarate and 1, v/v); system III for ketotifen hydrogen dimetindene maleate, 14 mm for promethazine fumarate: Merck HPTLC Silica gel 60 F254 chro- hydrochloride.

Figure 2. UV spectrum of azelastine hydrochloride measured directly from the TLC plate

Figure 3. UV spectrum of dimetindene maleate measured directly from the TLC plate

Figure 4. UV spectrum of promethazine hydrochloride measured directly from the TLC plate 956 ELØBIETA WYSZOMIRSKA et al. [%] RSD [%] RSD X ∆ X ∆ [%] S [%] Arithmetic mean of Standard Confidence interval X ± all measurements X deviation PU = 95% samples X S PU = 95% Number Arithmetic mean of Standard Confidence interval 6 100.33 0.11 100.33 ± 0.10 0.10 7 7 99.78 100.73 0.30 0.16 99.78 ± 0.33 100.73 ± 0.17 0.28 0.16 Number of HPLC HPLC HPLC 7 7 6 0.99 mg/mL 0.95 mg/g 0.00 25.95 mg 0.99 mg/mL ± 0.00 mg 0.01 0.28 0.54 0.95 mg/g ± 0.01 mg 25.95 mg ± 0.52 1.16 2.10 Method ) HPLC 6 0.98 mg 0.01 0.98 mg ± 0.01 1.41 Titration method anhydrous medium anhydrous medium Titration method in Titration method in in anhydrous medium Densitometric method 7 99.70 0.31 99.70 ± 0.44 0.31 Densitometric method 6 100.38 0.14 100.38 ± 0.14 0.14 Densitometric method 7 100.18 0.61 100.18 ± 0.66 0.61 Tested compound Method of all measurements deviation X ± Fenistil 1 mg/g gel Densitometric method 7 0.97 mg/g 0.01 0.97 mg/g ± 0.02 mg 1.60 Tested maleate fumarate Ketotifen 1 mg tablets Densitometric method 6 0.97 mg 0.01 0.97 mg ± 0.01 1.28 Ketotifen hydrogen (Dimetindene maleate) Azelastine compound samples Allergodil 0.1% solution Densitometric method 7 1.00 mg/mL 0.01 1.00 mg/mL ± 0.01 mg 0.65 Diphergan 25mg dragÈes Densitometric method 6 25.83 mg 0.08 25.83 mg ± 0.10 0.32 (Azelastine hydrochloride) (Azelastine hydrochloride) Dimetindene hydrochloride Ketotifen hydrogen fumarate Ketotifen hydrogen (Promethazine hydrochloride) hydrochloride) (Promethazine ( Table 3. Statistical assessment of the results concerning determination tested compounds in pharmaceutical products. Table 2. Statistical results of the determination tested compounds in pharmaceutical substances. Identification and determination of ketotifen hydrogen fumarate... 957

Maximum wavelengths were determined for 0.1, 0.2, 0.3, 0.5 and 0.7 mg/mL for dimetindene all analyzed compounds: 228 nm for ketotifen maleate and Fenistil 1 mg/g gel; 0.25, 0.5, 0.75, 1 hydrogen fumarate (Fig. 1), 295 nm for azelastine and 1.25 mg/mL for promethazine hydrochloride hydrochloride (Fig. 2), 265 nm for dimetindene and Diphergan 25 mg dragÈes. maleate (Fig. 3) and 255 nm for promethazine All samples (excluding Allergodil 0.1% solu- hydrochloride (Fig. 4). tion) were shaken for 14 min in an ultrasonic bath and for 30 min in a mechanical shaker and strained Determination of regression curves through 0.45 µm filters. Portions of 10 mL of pre- Solutions of standards, substances (excluding pared solutions were transferred onto chromato- promethazine hydrochloride, as authors do not have graphic plates (2 cm from the edge and 2 cm from access to this substance) and medicinal products the bottom) and portions of: 0.2, 0.5, 1, 1.5, 2.5, 4 with following concentrations were prepared in and 5 µL were transferred directly from container of methanol: 0.5, 0.75, 1, 1.25 and 1.5 mg/mL for keto- Allergodil 0.1% solution. After the development (up tifen hydrogen fumarate and Ketotifen 1 mg tablets; to 1 cm from the top plate edge), the plates were air 0.02, 0.05, 0.1, 0.15, 0.25, 0.4 and 0.5 mg/mL for dried and examined in 254 nm UV light and densi- azelastine hydrochloride but Allergodil 0.1% solu- tometric measurements were made at selected wave- tion was transferred directly from container; 0.05, lengths determined prior to analysis.

Figure 5. Densitogram of ketotifen hydrogen fumarate at 228 nm Figure 6. Densitogram of azelasine hydrochloride at 295 nm

Figure 7. Densitogram of dimetindene maleate at 265 nm Figure 8. Densitogram of promethazine hydrochloride at 255 nm 958 ELØBIETA WYSZOMIRSKA et al.

The regression curves were determined for azelastine hydrochloride determination; system 5 is analyzed compounds, showing the linearity in the recommended to analyze azelastineís purity. following range: 5-15 µg/spot for ketotifen hydro- Systems 1 and 3 are the most suitable for determi- gen fumarate, 0.2-5 µg/spot for azelastine nation of dimetindene maleate. Systems 5 and 6 may hydrochloride, 0.5-7 µg/spot for dimetindene be used to analyze dimetindeneís purity. maleate, 2.5-12.5 µg/spot for promethazine Rf values and broadening of chromatographic hydrochloride. spots were taken into consideration when selecting Correlation coefficients R were as follows: system for quantitative analysis of specific compo- 0.9955 for ketotifen hydrogen fumarate, 0.9943 for nents. azelastine hydrochloride, 0.9937 for dimetindene Finally, the following systems were used for maleate, and 0.9925 for promethazine hydrochlo- quantitative analysis: system 2 for azelastine ride. Equations of a curve for specific compounds hydrochloride, dimetindene maleate (Rf = ca. 0.3 for were as follows: y = 9589.6x + 62560 for ketotifen both compounds) and promethazine hydrochloride hydrogen fumarate, y = 21475x + 12704 for azelas- (Rf = ca. 0.4), system 3 for ketotifen hydrogen tine hydrochloride, y = 17727x + 15920 for fumarate (Rf = ca. 0.6). dimetindene maleate, y = 18018x + 95204 for Automatic application was used to ensure the promethazine hydrochloride. highest precision of the densitometric method. The limits of quantification (LOQ) were deter- Conformity of spectra for analyzed substances, mined on the basis of signal-to-noise (S/N) ratio (at drugs and standard substances was demonstrated an S/N of 10 : 1) and were as follows: 5 mg/spot for and linearity for all analyzed compounds was ketotifen hydrogen fumarate, 0.2 µg/spot for azelas- observed. tine hydrochloride, 0.5 µg/spot for dimetindene Ketotifen hydrogen fumarate: a 10 µL portion maleate and 2.5 µg/spot for promethazine of standard solutions and prepared solutions were hydrochloride. Statistical data obtained for devel- applied on Merck HPTLC Silica gel 60 F254 chro- oped densitometric methods and comparative meth- matographic plates. ods are similar. Azelastine hydrochloride: a 1.5 µL portion of standard solutions, prepared solutions and Quantitative analysis Allergodil 0.1% solution (directly from container) Preparation of solutions: were transferred into Merck HPTLC Silica gel 60

The following concentrations of analyzed sub- F254 chromatographic plates. stances, standard substances and medicines (exclud- Dimetindene maleate: a 20 µL portion of stan- ing Allergodil 0.1% solution which was transferred dard solutions and prepared solutions were trans- directly from container) were prepared in methanol: ferred into Merck HPTLC Silica gel 60 F254 chro- 1 mg/mL for ketotifen hydrogen fumarate, promet- matographic plates. hazine hydrochloride and azelastine hydrochloride Promethazine hydrochloride: a 5 µL portion of and 0.1 mg/mL for dimetindene maleate. standard solutions and prepared solutions were

The samples were shaken for 14 min in an ultra- transferred into Merck HPTLC Silica gel 60 F254 sonic bath and for 30 min in a mechanical shaker. chromatographic plates. The solutions were strained through 0.45 µm filters. After the development (up to 1 cm from the top plate edge), the plates were air dried and densito- Determination of the content metric measurements at the selected wavelengths Several different mobile phases and different were made (Figs. 5ñ8). types of chromatographic plates were tested. The Simultaneously, determination of tested com-

Merck HPTLC Silica gel 60 F254 or Merck HPTLC pounds in medicinal products and substances using

LiChrospher Silica gel 60 RP-18 WF254s chromato- reference methods (described in Ph. Eur.) was con- graphic plates by Merck were chosen as stationary ducted. The titration method in anhydrous medium phases. was applied for determination of all substances. Of the seven chromatographic system tested, HPLC (described for the determination of impuri- based of the results, systems 2, 3 and 7 are the most ties) method was used for determination in medici- suitable for determination of ketotifen hydrogen nal products. fumarate and promethazine hydrochloride. Other The results and statistical assessment were pre- systems can be used to analyze these substancesís sented in Tables 2 and 3. purity. Systems 1, 3 and 4 are the most suitable for Identification and determination of ketotifen hydrogen fumarate... 959

CONCLUSIONS 6. Erny G.L., Cifuentes A.: J. Pharm. Biomed. Anal. 40, 509 (2006). The developed densitometric methods are sim- 7. Matysov· L., HavlÌkov· L., H·jkov· R., Krivda ple, fast and cost-effective. A., Solich P.: J. Pharm. Biomed. Anal. 50, 23 The comparative methods of the results (HPLC (2009). for products and titration method in anhydrous 8. MartÌnez-GÛmez M.A., Carril-AvilÈs M.M., medium for substances) showed that the deviations Sagrado S.: J. Chromatogr. A 1147, 261 (2007). in the results are statistically insignificant. New 9. Saif M.J., Anwar J.: Talanta 67, 869 (2005). methods may be widely applied or analysis of tested 10. Basavaiah K.: Farmaco 59, 315 (2004). compounds. 11. Lara F.J., Garcia-Campana A.M., Ales-Barrero F., Bosque-Sendra J.M.: Anal. Chim. Acta 535, REFERENCES 101 (2005). 12. WÛjciak-Kosior M., Skalska A., Matysik A.: J. 1. Zejc A., Gorczyca M.: Drug Chemistry Pharm. Biomed. Anal. 41, 286 (2006). (Polish). p. 398, PZWL, Warszawa 2004. 13. Saleh T.A., Abulkibash A.M., Ibrahim A.E.: 2. Kostowski W, Herman Z.S.: Pharmacology Saudi Pharm. J. 20, 155 (2012). (Polish). p. 194, PZWL, Warszawa 2010. 14. Zhang Q., Zhan X., Li C., Lin T., Li L., Yin X., 3. Fei N., Jiuru L.: Anal. Chim. Acta 592, 168 He N., Shi Y.: Int. J. Pharm. 302, 10 (2005). (2007). 15. Alizadeh T., Akhoundian M.: Electrochim. 4. Fujimaki K., Lee X.P., Kumazawa T., Sato J., Acta 55, 3477 (2010). Sato K.: Forensic Toxicol. 24, 8 (2006). 16. Huang M., Gao J., Zhai Z., Liang Q., Wang Y., 5. Heinemann U., Blaschke G., Knebel N.: J. Bai Y., Luo G.: J. Pharm. Biomed. Anal. 62, Chromatogr. B 793, 389 (2003). 119 (2012).

Received: 10. 10. 2012